Determining the presence of bacterial endospores by detecting the presence of pyridine-2,6-dicarboxylic acid (dipicolinic acid) has been used by those skilled in the art for some time. This compound comprises a significant portion of viable spores, and is otherwise rare in nature (R. Lundin and L. Sacks, “High-resolution solid-state 13C nuclear magnetic resonance of bacterial spores: Identification of the alpha-carbon signal of dipicolinic acid,” Appl. Environ. Microbiol., vol. 54, no. 4, pp. 923–928, 1988). Various analytical methods are used to detect dipicolinic acid to indicate the presence of spores, including derivative spectroscopy (A. Warth, “Determination of dipicolinic acid in bacterial spores by derivative spectroscopy,” Anal. Biochem., vol. 130, no. 2, pp. 502–505, 1983); intrinsic fluorescence (A. Alimova, A. Katz, H. E. Savage, M. Shah, G. Minko, D. V. Will, R. B. Rosen, S. A. McCormick and R. R. Alfano, “Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation,” Appl. Opt., vol. 42, no. 19, pp. 4080–4087, 2003); luminescence following the addition of lanthanide salts (D. L. Rosen, C. Sharpless and L. B. McGown, “Bacterial spore detection and determination by use of terbium dipicolinate photoluminescence,” Anal. Chem., vol 69, pp. 1082–1085, 1997); mass spectrometry (M. B. Beverly, K. J. Voorhees and T. L. Hadfield, “Direct mass spectrometric analysis of Bacillus spores,” Rapid Commun. Mass Spectrom., vol. 13, no. 23, pp. 2320–2326, 1999); Fourier-transform infrared spectroscopy (H. Y. Cheung, J. Cui and S. Sun, “Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination,” Microbiology, vol. 145, pp. 1043–1048, 1999); Raman spectroscopy (U.S. Pat. No. 6,040,191 and H. Shibata, S. Yamashita, M. Ohe and I. Tani, “Laser Raman spectroscopy of lyophilized bacterial spores,” Microbiol. Immunol., vol. 30, no. 4, pp. 307–313, 1986); and plasma chromatography coupled to gas chromatography (U.S. Pat. No. 6,672,133 B1).
Detection of endospores through the presence of calcium dipicolinate has been utilized in U.S. patents through detection of either the calcium and/or the dipicolinic acid. U.S. Pat. No. 6,498,041 B1 describes capture of spores based upon a molecular recognition of spore coat components followed by detection of Ca2+ by way of addition of fluorescent calcium-binding dyes excited by light in the visible spectrum. U.S. Pat. No. 6,599,715 and U.S. patent application Ser. No. 10/355,462 teaches detection of dipicolinic acid by way of luminescence from terbium dipicolinate when excited with ultraviolet light.
Furthermore, the presence of dipicolinic acid (or other pyridine dicarboxylic acid analogs with closely related chemical structures) has been reported for other cryptobiotic microorganisms. (Cryptobiotic describes microbes capable of achieving a dormant state). Specifically, dipicolinic acid has been utilized to detect Clostridium spores, (M. W. Tabor, J. MacGee and J. W. Holland, “Rapid determination of dipicolinic acid in the spores of Clostridium species by gas-liquid chromatography,” Appl. Environ. Microbiol., vol. 31, no. 1, pp. 25–28, 1976); Sporosarcina spores (C. A. Loshon and P. Setlow, “Levels of small molecules in dormant spores of Sporosarcina species and comparison with levels in spores of Bacillus and Clostridium species,” Can. J. Microbiol., vol. 39, no. 2, pp. 259–262, 1993); Sarcina spores (R. S. Thompson and E. R. Leadbetter, “On the isolation of dipicolinic acid from endospores of Sarcina ureae,” Arch. Mikrobiol., vol. 45, pp. 27–32, 1963); and Metabacterium spores (S. Stunkel, J. Alves and I. Kunstyr, “Characterization of two ‘Metabacterium’ sp. from the gut of rodents. Heteroxenic cultivation and proof of dipicolinic acid in ‘M. polyspora,’” Folia Microbiol. (Praha), vol. 38, no. 3, pp. 171–175, 1993). Pyridine dicarboxylic acid compounds are found in these and other cryptobiotic (spore-forming) microorganisms.
U.S. patent application Ser. No. 10/054,419, filed Jan. 22, 2002, and incorporated herein by reference, discloses a method and apparatus for the detection of microbes on non-living surfaces and samples in which samples are exposed to electromagnetic radiation of numerous specific energies capable of exciting fluorescence from various metabolites, cofactors and cellular and spore components. Thus, the microbial cells and spores to be sampled (and more specifically the excited metabolites, cofactors and other cellular, viral and/or spore components) contained therein emit fluorescence that can be measured. The collected fluorescence signals (associated with the signals emitted from the cellular/viral/spore components) are analyzed with a method capable of (1) removing any background and/or reflected and scattered excitation signal, and (2) comparing the relative fluorescent signals of metabolites, cofactors and spore components to known physiological ranges. Specifically, U.S. patent application Ser. No. 10/054,419 teaches the detection of spores by excitation of calcium dipicolinic acid with ultraviolet electromagnetic radiation (light) in the 270 nm–290 nm and 310 nm–330 nm ranges (singly or concurrently), with detection of fluorescence energies in the 460 nm–480 nm and 400 nm–430 nm regions, respectively. The aforementioned application also teaches the detection of spores by excitation with electromagnetic radiation (light) in the 610 nm–670 nm range with detection of light energies in the 730 nm–800 nm region. This novel emission was observed in emission spectra from aqueous bacterial spore samples and in a non-viable Bacillus thuringiensis cell sample as illustrated in FIG. 3F of the aforementioned application. Utilizing these novel lower energy excitation and emission ranges for the detection of spores is beneficial as (1) there is little interference and/or overlap from other microbial fluorophores, (2) background interference from biologically-derived organic surfaces is greatly reduced, and (3) greater excitation penetration depth into the sample can be expected. This current specification demonstrates that the beneficial lower energy excitation and emission signals arise from calcium dipicolinate and teaches the benefits of using these excitation sources for the detection of spores.
As is known to those skilled in the art, fluorescence is a form of luminescence. [Fluorescence and phosphorescence are defined as types of photoluminescence spectrometry (J. D. Ingle, Jr. and S. R. Crouch, Spectrochemical Analysis, pp. 438, 1988, Prentice-Hall, Inc.).] The primary difference between fluorescence and phosphorescence is the emission lifetimes (I. Tinoco, Jr., K. Sauer and J. C. Wang, Physical Chemistry: Principles and Applications in Biological Sciences, pp. 577, 1995, Prentice-Hall). (Fluorescence refers to emission lifetimes that are in the microsecond and shorter range; phosphorescence refers to emission lifetimes are typically in the millisecond or longer range.) Thus, without data of emission lifetimes, phosphorescence and fluorescence are experimentally indistinguishable using traditional emission spectroscopy. In this case, the ‘apparent fluorescence’ from the intrinsic chromophores (chemical components that absorb excitation energies and emit radiation of lower energy) may arise from either phosphorescence or fluorescence. Detection of apparent fluorescence from intrinsic microbial components confers the ability to detect dormant cryptobiotic microbes (1) without making physical contact with the sample, (2) very rapidly, and (3) without the use of any added reagents.
As can be readily appreciated, it would be very useful to be able to determine the presence of dormant (cyrptobiotic and/or spore-forming) microorganisms in hospitals, food preparation areas, water supplies, buildings and on the battlefield as these microbes require the greatest effort to eradicate. This method and apparatus, as an object of the invention, should be operated inexpensively and rapidly in, for example, food production facilities.